Oligonucleotide 5′-triphosphates (ONTPs) are not commercially available; still they find a great number of important biochemical applications: DNA ONTPs are used in the antisense field, in basic research or in the biotechnology industry (Brownlee, et al., Nucleic Acids Research 1995, 23, (14), 2641-2647)), as substrates for polymerases or ligases in the preparation of synthetic genes (Xiong, et al., Fems Microbiology Reviews 2008, 32, (3), 522-540); various synthetic RNA ONTPs can be used as primers for the amplification of RNA molecules by a 5′-pyrophosphate activated, template directed oligoribonucleotide ligation—either catalyzed by RNA ligases or non enzymatic; but also in detection of viral responses via activation of the RIG-I protein; induction of antiviral immunity (Joyce, et al., Angewandte Chemie-International Edition 2007, 46, (34), 6420-6436; Ekland, et al., Science 1995, 269, (5222), 364-370; Rohatgi, et al., Journal of the American Chemical Society 1996, 118, (14), 3340-3344; Hornung, et al., Science 2006, 314, (5801), 994-997; Allam, et al. Eur J Immunol 2008); or for their enzymatic conversion to 5′-capped RNAs (Brownlee, et al., Nucleic Acids Research 1995, 23, (14), 2641-2647); Olsen, et al. Journal of Biological Chemistry 1996, 271, (13), 7435-7439), the latter being useful for the determination of particular viral sequences, for the biochemical characterization of specific cap enzymes and the associated mRNA cap complexes, in order to study the translation mechanisms (Peyrane, et al., Nucleic Acids Research 2007, 35, (4)). Moreover, as recently reported (Poeck, et al., Nat Med 2008, 14, (11), 1256-63), the triggered immune response following 5′-triphosphate RNA binding to RIG-I synergized with oligonucleotide-mediated gene silencing, to cause massive apoptosis in tumor cells by using 5′-triphosphate oligonucleotide as a single molecule double-targeted treatment. This data can only suggest about the high therapeutic potential of immunostimulatory nucleic acids to be exploited in the future (Barchet et al., Curr Opin Immunol 2008, 20, (4), 389-95). In addition, very recent insights in the nature of the controversial RIG-I substrate-type could be brought into light thanks to the use of synthetic RNA ONTPs instead of the 5′-triphosphorylated products generated by in vitro RNA transcription (Ujita, et al. Immunity 2009, 31, (1), 4-5; Schlee et al., Immunity 2009, 31, (1), 25-34; Schmidt, et al., Proc Natl Acad Sci USA 2009, 106, (29), 12067-72).
There are several advantages in using synthetic 5′-triphosphate RNA over 5′-triphosphate RNA generated by in vitro transcription, those include: higher purity and clearer identity of the products which are obtained reproducibly and independently from the RNA sequences used; possibilities of scale-up synthesis and introduction of theoretically all the known RNA chemical modifications (Atts, et al., Drug Discovery Today 2008, 13, (19-20), 842-855).
Despite these numerous applications and advantages, DNA and RNA ONTPs are difficult for access, as there is no easy and efficient method for their enzyme free, chemical synthesis. Hence, the chemical preparation of ONTPs seems to be a real challenge, since the few known recent approaches describing their synthesis on solid support (Lebedev, et al., Nucleosides Nucleotides Nucleic Acids 2001, 20, (4-7), 1403-9) are all associated with low efficiency, serious lack of universality in regards of the length and the sequence, difficult separation procedures resulting from low conversions, and eventually poor yields. The polyfunctional oligomeric nature of the RNA or DNA substrate, which involves the precise choice of appropriate protecting groups and overall synthetic strategy, can only be added to the existing limitations known for nucleoside triphosphate (NTP) synthesis (Burgess, et al., Chemical Reviews 2000, 100, (6), 2047-2059.). Moreover, as witnessed by several recent reports, synthetic efforts are still ongoing for developing a simple, efficient and universal triphosphorylation method for nucleosides (Crauste, et al., The Journal of Organic Chemistry 2009, 74, 9165-9172; Sun, Q et al., Organic Letters 2008, 10, (9), 1703-1706; Warnecke, et al., The Journal of Organic Chemistry 2009, 74, (8), 3024-3030).